Addition of ticarcillin-clavulanic acid to INRA96 extender in the extension and storage of equine semen: An examination of its effect on semen motion characteristics and viability

The use of stallion semen extenders containing antimicrobials is increasingly common in equine breeding facilities. Breeders now have numerous options for commercially available semen extenders for use in fresh, cooled semen. Commercially produced semen extenders, such as INRA 96®, commonly contain antimicrobials that may have limited efficacy. When this is a concern, many breeding facilities in the United States rely on the addition of ticarcillin-clavulanic acid to inhibit or eliminate possible bacterial growth. This practice, however, needs standardization from proven data.

The focus of this study was on evaluating the effects of the addition of ticarcillin-clavulanic acid, Timentin®, to INRA 96. This included assessing the effect of different extender and antimicrobial storage conditions on sperm motion characteristics, sperm membrane integrity and antimicrobial effectiveness. Fourteen mature, actively breeding Quarter Horse stallions were collected and 42 gel-free semen ejaculates were diluted with INRA 96 extender and stored for 24 hours in Equitainer II units.

Post storage evaluations consisted of sperm motion characteristics analysis by a computer-assisted analysis device, sperm membrane integrity evaluation by fluorescent measurements and bacterial isolation cultures for both aerobic and anaerobic bacteria. Data evaluation included analysis-of-variance and chi-square statistical methods with the P value for significance predetermined to < 0.05.

Significant reduction in sperm motion characteristics, total motility, progressive motility and sperm membrane integrity, after 24 h of cooled storage were found in treatments that were subjected to freezing and thawing of modified or unmodified extender prior to use. The addition of reconstituted ticarcillin-clavulanic acid to the extender prior to use resulted in higher sperm velocity when treatments were exposed to cooled storage rather than frozen. Bacterial isolates were cultured from neat semen in only 28 of 42 ejaculates (67%). The addition of ticarcillin-clavulanic acid to INRA 96 was no different than the use of INRA 96 alone for the inhibition of bacterial growth (98% vs. 94%).

The addition of ticarcillin-clavulanic acid at a dose rage of 1 mg/ml to INRA 96 extender did not negatively impact sperm motion characteristics and viability in extended semen after cooled storage. The act of freezing and thawing of extender prior to use did however have negative effects on sperm quality. This trial was conducted on a commercial breeding facility that did not have any cases of Pseudomonas aeruginosa or Klebsiella pneumoniae. Thus, additional studies are needed using samples that could be subjected to such pathogens.

Results of this study suggest that INRA 96 extender should not be exposed to freezing and thawing prior to use. Bacterial cultures of the extender treatments indicated effective inhibition of bacterial growth when compared to neat semen.